A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

Summary

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Abstract

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a "seed coat bedding" assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

Introduction

In Arabidopsis mature seeds, the seed coat is composed of the testa, an external layer of dead tissue of maternal origin, and the endosperm, a single cell layer of live tissue directly surrounding the embryo¹. The endosperm and the embryo are derived from separate fertilization events: the endosperm is a triploid tissue with two maternal and one paternal genome whereas the embryo is a diploid tissue with one maternal and one paternal genome².

The main function traditionally assigned to the endosperm is that of a nutritive tissue. However, it is becoming increasingly evident that the endosperm also plays a cent....

Protocol

Once the SCBA is assembled, the growth of embryos is monitored over several days. Therefore, before the seed dissection procedure and assembly of the SCBA, one needs to sterilize seeds to avoid future contaminations that could prevent proper assessment of the effect of seed coat material on embryonic growth.

1. Seed Sterilization

1. Pour 50-60 μl of mature and dry Arabidopsis seeds in a 1.5 ml microcentrifuge tube and prepare a 70% ethanol solution.
2. Add 1 ml of 70% ethanol solution to the microcentrifuge tube containing the seeds and shake at room temperature for 10 min at 1,200 rpm in a vortexer.
3. ....

Results

Previous work showed that mutant seeds unable to synthesize GA were unable to germinate as a result of high ABA accumulation in seeds ^11,12. However, inability to germinate requires the seed coat since its removal triggers embryonic growth¹³. This strongly indicated that the endosperm of seeds unable to synthesize GA is releasing ABA to block embryonic growth. We therefore expect seeds coats unable to synthesize GA to block the growth of embryos in a SCBA unlike seed coats unable to synthesize GA an.......

Discussion

The seed coat bedding assay (SCBA) procedure described here is in principle applicable to any circumstance where Arabidopsis seed germination is blocked (or delayed) and where the endosperm is suspected to implement this arrest. The latter can be evidenced by removing the seed coat (testa and endosperm) and observing that embryonic growth proceeds faster relative to that observed when embryos are surrounded by the seed coat. Germination may be blocked in response to particular environmental physical parameters (.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thermomixer Comfort	Eppendorf AG	5355 000.011	Eppendorf AG, Hamburg, Germany
Vacusafe Comfort	INTEGRA Biosciences AG	158 310	Integra Biosciences AG, Zizers, Switzerland
Petri dish plate (100 mm x 20 mm)	Greiner Bio-One GmbH	664 102	Greiner Bio-One GmbH, Frickenhausen, Germany
Murashige and Skoog	Sigma-Aldrich	M5524	Sigma-Aldrich, St Louis, MO, USA
MES	Sigma-Aldrich	M3671	Sigma-Aldrich, St Louis, MO, USA
Agar (plant agar)	Duchefa Biochemie B.V.	P1001	Duchefa Biochemie, Haarlem, Netherlands
Dumont forceps #5	Fine Science Tools GmbH	11251-10	Fine Science Tools GmbH, Heidelberg Germany
Syringe needle	BD Micro-Fine	324827	BD, Franklin Lakes, NJ USA
Nylon mesh (SEFAR NYTEX)	SEFAR AG	03-50/31	Sefar AG, Heiden, Switzerland
Growth chamber	CLF Plant Climatics	Percival I-30BLLX	CLF plant Climatics, Wertingen, Germany
Paclobutrazol	Sigma-Aldrich	46046	Sigma-Aldrich, St Louis, MO, USA